As polymorphisms of genes involving obesity, polymorphisms of the gene coding for β-2 adrenaline receptor (β2AR) (β2AR gene), the gene coding for β-3 adrenaline receptor (β3AR) (β3AR gene), and the gene coding for uncoupling protein (UCP) (UCP1 gene) are known. β2AR is mainly distributed in the heart and the bronchial smooth muscle. Besides them, β2AR is present in fat tissue and is involved in lipolysis. Further, the β2AR gene coding for β2AR includes a polymorphism (Arg16Gly) in which arginine (Arg) at position 16 of amino acid is changed to glycine (Gly). It has been reported that, as compared to subjects not having the aforementioned polymorphism, a resting metabolism amount of subjects having this polymorphism (16% in Japanese) is increased. Further, β3AR is present in brown adipose tissue and white adipose tissue. Upon binding of noradrenaline, thermogenesis in the brown adipose tissue and lipolysis in the white adipose tissue are started, respectively. The β3AR gene coding for β3AR includes a polymorphism (Trp61Arg) in which tryptophan (Trp) at position 64 of amino acid is changed to arginine (Arg). It has been reported that, as compared to subjects not having the aforementioned polymorphism, a resting metabolism amount of subjects having this polymorphism (34% in Japanese) is decreased. Further, UCP1 is a protein that plays a main role in the thermogenesis in the brown adipose tissue when sympathetic nerve is in an excited state. The UCP1 gene coding for UCP1 includes a polymorphism in which adenine CA) at position −3826 in mRNA (position 3826 at upstream of a translation initiation site of the UCP1 gene in the genome sequence) is changed to guanine (G). It has been reported that, as compared to subjects not having the aforementioned polymorphism, a total body metabolism amount of subjects having this polymorphism (24% in overweight Japanese female) is decreased. On the bases of the relationship between polymorphisms of the aforementioned respective genes and metabolism amounts, the polymorphisms of these genes are analyzed. Analysis results thereof are practically used for treatment and prevention of obesity of the subjects.
On the other hand, detection of a point mutation, a so-called single nucleotide polymorphism (SNP), is employed widely as an analysis method, at the gene level, for example, the causes of all types of diseases and the individual differences in disease liability (susceptibility to diseases) and in drug action. Examples of the common methods of detecting a point mutation include: (1) a direct sequencing method in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by a polymerase chain reaction (PCR) and all the gene sequences are analyzed, (2) a RFLP analysis in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by PCR, the amplification product thus obtained is cut with a restriction enzyme whose cleaving action differs depending on the presence or absence of the target mutation in the sequence to be detected and is then electrophoresed, and thereby typing is performed, and (3) the ASP-PCR method in which PCR is performed using a primer with a target mutation located at the 3′-end region and the mutation is judged depending on the presence or absence of amplification.
However, since these methods require, for example, purification of DNA extracted from a sample, electrophoresis, and a treatment with a restriction enzyme, they take time and cost. Furthermore, after PCR is performed, it is necessary to open the reaction container once. Accordingly, there is a possibility that the amplification product may contaminate the next reaction system and thereby the analysis accuracy may be deteriorated. Moreover, since it is difficult to automate, multiple samples cannot be analyzed. Further, the aforementioned ASP-PCR method (3) is less specific, which also is a problem.
Because of these problems, recently, a method of analyzing the melting temperature (Tm) of double-stranded nucleic acid formed of a probe and target nucleic acid is used as a method of detecting a point mutation. Since such a method is performed through, for example, Tm analysis or analysis of the melting curve of the double strand, it is referred to as melting curve analysis. This method is described below. That is, first, a probe complementary to a sequence to be detected containing a target point mutation is used to form a hybrid (double-stranded DNA) between the aforementioned probe and a target single-stranded DNA contained in a detection sample. Subsequently, this hybridization product is heat-treated, and dissociation (melting) of the hybrid accompanying the temperature rise is detected by a change in a signal such as absorbance. The Tm value is then determined based on the result of the detection and the presence or absence of any point mutation is judged accordingly. The higher the homology of the hybridization product, the higher the Tm value, and the lower the homology, the lower the Tm value. Therefore the Tm value (reference value for assessment) is determined beforehand with respect to the hybridization product between the sequence to be detected containing a point mutation and a probe complementary thereto, and then the Tm value (measured value) of the hybridization product between the target single-stranded DNA contained in the detection sample and the aforementioned probe is measured. When the measured value is comparable to the reference value, it is considered as matching, that is, it can be judged that a point mutation is present in the target DNA. On the other hand, when the measured value is lower than the reference value, it is considered as mismatching, that is, it can be judged that no point mutation is present in the target DNA. Furthermore, according to this method, it also is possible to automate the gene analysis.
However, such a detection method using Tm analysis also has a problem in that a region including a site to be detected must be able to be amplified specifically and efficiently in PCR. Particularly, many isozymes are present in the β2AR gene, the β3AR gene, and the UCP1 gene, which are important genes among the obesity genes, and the sequences for coding them also are very similar to one another. Accordingly, there is a possibility that genes coding for isozymes other than those desired genes also are amplified in PCR. Furthermore, when other isozyme-coding genes also have been amplified as described above, for example, in analysis in each polymorphism of the β2AR gene, the β3AR gene, and the UCP1 gene (Nonpatent Documents 1-3), it may cause a decrease in reliability of the analysis result. Moreover, as described above, since analysis of one sample is accompanied by a considerable amount of time and energy, it is not practical to analyze multiple samples, which also is a problem.    [Nonpatent Document 1] PMID: 9399966 J Clin Invest. Dec. 15, 1997; 100(12): 3184-8.    [Nonpatent Document 2] PMID: 9049481 Diabetologia. February 1997; 40(2): 200-4.    [Nonpatent Document 3] PMID: 9541178 Diabetologia. March 1998; 41(3): 357-61.